PB2509: RED BLOOD CELL PYRUVATE KINASE PROPERTIES IN SICKLE CELL DISEASE – OF MICE AND MEN
Authors: Marissa Traets, Brigitte van Oirschot, Charles Levine, Judith Jans, Minke Rab, Yu-Wei Chen, Richard Van Wijk
Journal: HemaSphere (2023)

Topic: 26. Sickle cell disease Background: Townes and Berkeley mouse models of sickle cell disease (SCD) are commonly used to study disease pathophysiology and to develop novel therapies. Despite similar pathophysiology to SCD patients, there are distinctive differences. For example, Townes HbSS mice have higher ATP and lower 2,3-diphosphyglycerate (2,3-DPG) levels compared to HbAA mice. This contrasts with SCD patients, who show high 2,3-DPG but low ATP, suggesting potential differences between human and murine red blood cell (RBC) metabolism. Pyruvate kinase (PK), a key ATP-generating enzyme in glycolysis, is a target for novel SCD therapies. While PK activators have been shown to increase hemoglobin in clinical trials, such treatments do not always lead to the same hematological change in mouse models, further suggesting that PK features may differ between SCD mouse models and human patients. A detailed characterization of PK and related important cellular metabolites in murine SCD models and SCD patients is therefore important. Aims: To study PK features in murine and human SCD RBCs. Methods: Wild-type (C57BL/6J), Townes and Berkeley mice (non-sickling and sickling) were studied. Human blood was obtained from healthy controls (HbAA) and untreated SCD patients (HbSS). Hematological parameters were measured using the Cell-Dyn Sapphire (Abbott Diagnostics). PK and hexokinase (HK) activity, and PK thermostability (53°C) were measured on purified RBCs. Levels of ATP, 2,3-DPG, and reduced glutathione (GSH) were measured by LC-MS/MS and spectrophotometry, respectively. Results: Samples from 12 wild-type mice, 13 Townes mice (6 HbAA, 7 HbSS), 6 Berkeley mice (3 non-sickling, 3 sickling) and 10 human subjects (6 HbAA, 4 HbSS) were included. High reticulocyte counts compared to control mice were observed in both Townes and Berkeley SCD mice (median 59% and 48% compared to 7% and 5%, respectively). This was accompanied by an increased activity of PK in both SCD models (Figure 1A). When normalizing PK activity to that of HK, another age-dependent enzyme, no difference was found between sickling and non-sickling mice for both mouse models (Figure 1B). However, PK/HK ratio was lower in the Berkeley mice compared to Townes mice (p<0.01 for both non-sickling and sickling mice). ATP/2,3-DPG ratio was higher in Townes and Berkeley sickling mice compared to their control mice (Figure 1C; p<0.01). This contrasts with humans where healthy controls had higher ATP/2,3-DPG ratio than SCD patients (p<0.05). Surprisingly, mouse RBC PK was considerably less stable than its human counterpart, as reflected by a strikingly decreased thermostability (Figure 1D). GSH levels were significantly increased in both Townes and Berkeley sickling mice compared to non-sickling mice, which is in contrast with previous findings that SCD patients have lower GSH levels. Summary/Conclusion: PK/HK ratios are similar between sickling and non-sickling mice in both Berkeley and Townes model strains. In both models, however, there are significant changes in energy level (ATP), 2,3-DPG (important for oxygen affinity), and antioxidant level (GSH) when compared sickling to non-sickling mice. Our results further reveal important distinctions between mouse models and humans: the lack of PK/HK ratio difference between sickling and non-sickling mice, and overall lower PK thermostability in mice. Sickling mice also have higher ATP/2,3-DPG ratio and GSH levels, which is very different from SCD patients. These differences should be considered when studying PK activators in SCD mouse models and extrapolating results to a clinical response.Keywords: Transgenic mice, Pyruvate kinase, Sickle cell disease